Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further.

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An A/U-Rich Enhancer Region Is Required for High-Level Protein Secretion through the HlyA Type I Secretion System

Applied and Environmental Microbiology ◽

10.1128/aem.01163-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 3

Author(s):

Sakshi Khosa ◽

Romy Scholz ◽

Christian Schwarz ◽

Mirko Trilling ◽

Hartmut Hengel ◽

...

Keyword(s):

Ribosomal Protein ◽

Protein Secretion ◽

Fusion Proteins ◽

Untranslated Region ◽

Secretion System ◽

Type I ◽

Secretion Systems ◽

Enhancer Region ◽

Ribosomal Protein S1 ◽

Cellular Expression

ABSTRACTEfficient protein secretion is often a valuable alternative to classic cellular expression to obtain homogenous protein samples. Early on, bacterial type I secretion systems (T1SS) were employed to allow heterologous secretion of fusion proteins. However, this approach was not fully exploited, as many proteins could not be secreted at all or only at low levels. Here, we present an engineered microbial secretion system which allows the effective production of proteins up to a molecular mass of 88 kDa. This system is based on the hemolysin A (HlyA) T1SS of the Gram-negative bacteriumEscherichia coli, which exports polypeptides when fused to a hemolysin secretion signal. We identified an A/U-rich enhancer region upstream ofhlyArequired for effective expression and secretion of selected heterologous proteins irrespective of their prokaryotic, viral, or eukaryotic origin. We further demonstrate that the ribosomal protein S1 binds to thehlyAA/U-rich enhancer region and that this region is involved in the high yields of secretion of functional proteins, like maltose-binding protein or human interferon alpha-2.IMPORTANCEA 5′ untranslated region of the mRNA of substrates of type I secretion systems (T1SS) drastically enhanced the secretion efficiency of the endogenously secreted protein. The identification of ribosomal protein S1 as the interaction partner of this 5′ untranslated region provides a rationale for the enhancement. This strategy furthermore can be transferred to fusion proteins allowing a broader, and eventually a more general, application of this system for secreting heterologous fusion proteins.

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Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems

Journal of Molecular Biology ◽

10.1006/jmbi.1998.1791 ◽

1998 ◽

Vol 279 (3) ◽

pp. 485-499 ◽

Cited By ~ 158

Author(s):

Marjorie Russel

Keyword(s):

Protein Secretion ◽

Bacterial Cell ◽

Cell Envelope ◽

Type Ii ◽

Secretion Systems ◽

Macromolecular Assembly ◽

Bacterial Cell Envelope ◽

Protein Secretion Systems

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Simple and Minimally Invasive SID Devices for Native Mass Spectrometry

10.26434/chemrxiv.12145455.v1 ◽

2020 ◽

Author(s):

Dalton Snyder ◽

Erin Panczyk ◽

Arpad Somogyi ◽

Desmond Kaplan ◽

Vicki Wysocki

Keyword(s):

Minimally Invasive ◽

Ion Mobility ◽

First Generation ◽

Dynamic Range ◽

Protein Complexes ◽

Mass Range ◽

Native Mass Spectrometry ◽

Order Of Magnitude ◽

Sensitivity Improvement ◽

Ft Icr

We describe a set of simple devices for surface-induced dissociation of protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few mm along the ion path axis) and easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. The split lens used for SID is an order of magnitude smaller than the first-generation SID devices developed in our laboratory and approximately 5x smaller than the second-generation devices developed on the Q-IM-TOF and FT-ICR. Despite the decrease in size and reduction in number of electrodes to 3 (from 10-12 in Gen 1 and ~6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

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Simple and Minimally Invasive SID Devices for Native Mass Spectrometry

10.26434/chemrxiv.12145455 ◽

2020 ◽

Author(s):

Dalton Snyder ◽

Erin Panczyk ◽

Arpad Somogyi ◽

Desmond Kaplan ◽

Vicki Wysocki

Keyword(s):

Minimally Invasive ◽

Ion Mobility ◽

First Generation ◽

Dynamic Range ◽

Protein Complexes ◽

Mass Range ◽

Native Mass Spectrometry ◽

Order Of Magnitude ◽

Sensitivity Improvement ◽

Ft Icr

We describe a set of simple devices for surface-induced dissociation of protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few mm along the ion path axis) and easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. The split lens used for SID is an order of magnitude smaller than the first-generation SID devices developed in our laboratory and approximately 5x smaller than the second-generation devices developed on the Q-IM-TOF and FT-ICR. Despite the decrease in size and reduction in number of electrodes to 3 (from 10-12 in Gen 1 and ~6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

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Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase

mBio ◽

10.1128/mbio.01115-21 ◽

2021 ◽

Author(s):

Steven J. Hersch ◽

Linh Lam ◽

Tao G. Dong

Keyword(s):

Protein Secretion ◽

Gene Editing ◽

Cre Recombinase ◽

Secretion System ◽

Eukaryotic Cells ◽

Secretion Systems ◽

Protein Secretion System ◽

And Function

Delivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function.

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The Brucella suis Type IV Secretion System Assembles in the Cell Envelope of the Heterologous Host Agrobacterium tumefaciens and Increases IncQ Plasmid pLS1 Recipient Competence

Infection and Immunity ◽

10.1128/iai.74.1.108-117.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 108-117 ◽

Cited By ~ 13

Author(s):

Anna Carle ◽

Christoph Höppner ◽

Khaled Ahmed Aly ◽

Qing Yuan ◽

Amke den Dulk-Ras ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Mammalian Cells ◽

Cell Envelope ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Heterologous Host ◽

Type Iv ◽

Brucella Suis

ABSTRACT Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related α2-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.

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Simple and Minimally Invasive SID Devices for Native Mass Spectrometry

10.26434/chemrxiv.12145455.v2 ◽

2020 ◽

Author(s):

Dalton Snyder ◽

Erin Panczyk ◽

Arpad Somogyi ◽

Desmond Kaplan ◽

Vicki Wysocki

Keyword(s):

Minimally Invasive ◽

Ion Mobility ◽

First Generation ◽

Dynamic Range ◽

Protein Complexes ◽

Mass Range ◽

Native Mass Spectrometry ◽

Order Of Magnitude ◽

Sensitivity Improvement ◽

Ft Icr

We describe a set of simple devices for surface-induced dissociation of protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few mm along the ion path axis) and easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. The split lens used for SID is an order of magnitude smaller than the first-generation SID devices developed in our laboratory and approximately 5x smaller than the second-generation devices developed on the Q-IM-TOF and FT-ICR. Despite the decrease in size and reduction in number of electrodes to 3 (from 10-12 in Gen 1 and ~6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

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Negative Regulation of hrp Genes inPseudomonas syringae by HrpV

Journal of Bacteriology ◽

10.1128/jb.180.17.4532-4537.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4532-4537 ◽

Cited By ~ 74

Author(s):

Gail Preston ◽

Wen-Ling Deng ◽

Hsiou-Chen Huang ◽

Alan Collmer

Keyword(s):

Protein Secretion ◽

Constitutive Expression ◽

Secretion System ◽

Negative Regulator ◽

Wild Type ◽

Secretion Systems ◽

Type Iii ◽

Regulatory Cascade ◽

Group I ◽

Type Iii Protein Secretion

ABSTRACT Mutations in the five hrp and hrc genes in the hrpC operon of the phytopathogen Pseudomonas syringae pv. syringae 61 have different effects on bacterial interactions with host and nonhost plants. The hrcC gene within the hrpC operon encodes an outer membrane component of the Hrp secretion system that is conserved in all type III protein secretion systems and is required for most pathogenic phenotypes and for secretion of the HrpZ harpin to the bacterial milieu. The other four genes (in order), hrpF, hrpG, (hrcC), hrpT, and hrpV, appear to be unique to the group I hrp clusters found in certain phytopathogens (e.g., P. syringae and Erwinia amylovora) and are less well understood. We initiated an examination of their role in Hrp regulation and secretion by determining the effects of functionally nonpolar nptIIcartridge insertions in each gene on the production and secretion of HrpZ, as determined by immunoblot analysis of cell fractions. P. syringae pv. syringae 61 hrpF, hrpG, andhrpT mutants were unable to secrete HrpZ, whereas thehrpV mutant overproduced and secreted the protein. This suggested that HrpV is a negative regulator of HrpZ production. Further immunoblot assays showed that the hrpV mutant produced higher levels of proteins encoded by all three of the majorhrp operons tested—HrcJ (hrpZ operon), HrcC (hrpC operon), and HrcQB (hrpUoperon)—and that constitutive expression of hrpV intrans abolished the production of each of these proteins. To determine the hierarchy of HrpV regulation in the P. syringae pv. syringae 61 positive regulatory cascade, which is composed of HrpRS (proteins homologous with ς54-dependent promoter-enhancer-binding proteins) and HrpL (alternate sigma factor), we tested the ability of constitutively expressed hrpV to repress the activation of HrcJ production that normally accompanies constitutive expression of hrpL or hrpRS. No repression was observed, indicating that HrpV acts upstream of HrpRS in the cascade. The effect of HrpV levels on transcription of thehrpZ operon was determined by monitoring the levels of β-glucuronidase produced by ahrpA′::uidA transcriptional fusion plasmid in different P. syringae pv. syringae 61 strains. The hrpV mutant produced higher levels of β-glucuronidase than the wild type, a hrcU (type III secretion) mutant produced the same level as the wild type, and the strain constitutively expressing hrpV in trans produced low levels equivalent to that of a hrpS mutant. These results suggest that HrpF, HrpG, and HrpT are all components of the type III protein secretion system whereas HrpV is a negative regulator of transcription of the Hrp regulon.

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Characterisation of a highly sensitive troponin I assay and its application to a cardio-healthy population

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2011-0540 ◽

2012 ◽

Vol 50 (5) ◽

Cited By ~ 79

Author(s):

Gus Koerbin ◽

Jill Tate ◽

Julia M. Potter ◽

Juleen Cavanaugh ◽

Nicholas Glasgow ◽

...

Keyword(s):

Troponin I ◽

Dynamic Range ◽

Matrix Effects ◽

Healthy Population ◽

Distribution Of Values ◽

Highly Sensitive ◽

Order Of Magnitude ◽

Highly Correlated ◽

Highly Sensitive Troponin ◽

Apparently Healthy

AbstractAbbott Diagnostics have developed a new highly sensitive troponin I (hs-TnI) assay. We have assessed its analytical characteristics and applied the assay to a population of apparently cardio-healthy persons.We assessed imprecision, bias compared to the previous generation assay, matrix effects, and interferences and applied the assay to an apparently healthy population, deriving the 99th percentile limit of the distribution of values in reference populations for men and women separately.The dynamic range of the assay was ranged from 0.5–50,000 ng/L (pg/mL). The 10% CV was at a concentration of 3.9 ng/L, and the 20% CV was at a concentration of 1.8 ng/L. The new and current version of the TnI assay were highly correlated [slope: 0.98 (95%CI:0.88–1.07),This new hs-TnI assay is able to measure to an order of magnitude lower than the current generation TnI assay from the same manufacturer. With TnI being detectable in nearly all apparently healthy subject samples this suggests that TnI presence does not always indicate cardiomyocyte necrosis.

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